Antimicrobial and anticancer lipopeptides

ABSTRACT

The invention provides lipophilic conjugates comprising a peptide coupled to a fatty acid, the peptide comprising at least two positively charged amino acid residues, said peptide after conjugation to the fatty acid possessing antibacterial, antifungal, and/or anticancer activity higher than prior to conjugation. The lipophilic conjugates are suitable for treatment of infections caused by pathogenic organisms such as bacteria and fungi. The lipophilic conjugates are also suitable for sanitation, as disinfectants, or for food preservation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/560,727filed Oct. 10, 2006, which is the U.S. National Stage of Application No.PCT/IL2004/000544 filed Jun. 18, 2004, which in turn claims the benefitof Application No. 60/479,465 filed Jun. 19, 2003.

FIELD OF THE INVENTION

The present invention relates to lipophilic conjugates comprising fattyacids coupled to L-amino acid peptides, D-amino acid peptides ordiastereomeric peptides, to pharmaceutical compositions comprising same,and uses thereof in the treatment of infections and cancer diseases.

BACKGROUND OF THE INVENTION

The frequency of opportunistic fungal infections has increased in thelast decade. Invasive mycoses have emerged as major causes of morbidityand mortality (Groll, A. H. et al. (1996) J. Infect. 33: 23-32;Minamoto, G. Y. and Rosenberg, A. S. (1997) Med. Clin. North Am. 81:381-409; Walsh, T. J., et al. (1996) Infect. Dis. Clin. North Am. 10:365-400). The vast majority of the invasive fungal infections are causedby Aspergillus and Candida species (Denning, D. W. (1991) J. Antimicrob.Chemother. 28: 1-16). As fungal cells have a restricted set of specificmetabolic pathways and because of their eukaryotic nature, selectivetargeting of fungal cells has not been successfully achieved. Azolesthat inhibit sterol formation and polyenes that bind to mature membranesterols have been the mainstays of antifungal therapy for two decades ormore. However, the development of fluconazole resistance among differentpathogenic strains and the high toxicity of amphotericin B (Alexander,B. D. and Perfect, J. R. (1997) Drugs 54: 657-678) have prompted thesearch for new antifungal agents that can augment or replace therapeuticstrategies for mycotic infections in the near future.

The investigation of antimicrobial peptides from a wide range ofbiological sources, and their synthetic derivatives, is a novel approachto new antifungal agents. Antimicrobial peptides are part of the innateimmunity against microbial invasion in all organisms including human andplants and their possible mode of action has been reviewed in detail(Bechinger, B. (1999) Biochem. Biophys. Acta 1462: 157-183; Tossi, A.,et al. (2000) Biopolymers 55: 4-30; Oren, Z. and Shai, Y. (1998)Biopolymers 47: 451-463). It is believed that most of these peptidesbind and permeate the cell membranes. Antimicrobial peptides can beclassified into two groups: (i) cell selective antimicrobial peptidesthat act on a narrow spectrum of microorganisms such as bacteria orfungi, and (ii) non-cell selective antimicrobial peptides that can lyseboth microorganisms and normal mammalian cells.

One group of non-cell selective antimicrobial peptides includes nativelipopeptides. These lipopeptides possess a broad spectrum of activitiesincluding antibacterial, antifungal, antiviral and cytolytic. Some ofthese lipopeptides are gene-encoded and synthesized ribosomally.However, some are synthesized non-ribosomally in bacteria, yeast, orfungi during cultivation on various carbon sources.

U.S. Pat. No. 6,183,736 describes the identification and purification oftwo lipopeptides from the culture medium of the bacteria Bacillussubtilis. Both lipopeptides are cyclic, acidic, and have a broad rangeof antifungal and antibacterial activity. It is disclosed that the twolipopeptides can be used for controlling aflatoxin-producing fungicontamination in plants. U.S. Pat. No. 6,384,013 discloses otherantifungal lipopeptides, produced by culturing microorganisms. These arecyclic hexapeptides to which unique fatty acid acyl groups are attached.As a result of deacylation of the native acyl groups and reacylationwith the unique acyl groups, the peptides exert enhanced antifungal andantiparasitic potency against pathogenic strains of fungi.

Recently, the present applicants incorporated several D-amino acids inthe α-helical cytolytic peptides pardaxin and melittin (U.S. Pat. No.6,172,038 and Shai, Y. and Oren, Z. (1996) J. Biol. Chem. 271:7305-7308; Oren, Z., and Shai, Y. (1997) Biochemistry 36: 1826-1835).The resulting peptides of pardaxin and melittin having both L- andD-amino acids retained high antibacterial activity, while exhibitingreduced cytotoxic effects in mammalian cells. These peptides paved theway for the design of novel peptide antibiotics comprising both D- andL-amino acids that are selective to microorganisms.

U.S. Pat. No. 6,172,038 and WO 98/37090 disclose non-natural syntheticpeptides comprising both L- and D-amino acid residues designateddiastereomeric peptides with a net positive charge that is greater than+1. Some synthetic peptides consist of at least one hydrophobic aminoacid and at least one positively charged amino acid, in which at leastone of the amino acid residues is a D-amino acid. Several diastereomericpeptides containing from 6 to 30 amino acid residues are disclosed inU.S. Pat. No. 6,172,038 and WO 98/37090. Certain 12-mer peptides thatcontain lysine and a hydrophobic amino acid, in which one-third of theamino acid residues of the peptide are D-amino acid residues, werefurther investigated and found to be potent antimicrobial peptideshaving reduced hemolytic activity (Oren, Z., et al. (1997) J. Biol.Chem. 272: 14643-14649; Hong, J., et al. (1999) Biochemistry 38:16963-16973; Avrahami, D., et al. (2001) Biochemistry 40: 12591-12603).WO 02/40529 discloses additional diastereomeric peptides havingantibacterial, antifungal and anti-cancer activity.

In some studies investigators attached fatty acids to antimicrobialpeptides in order to improve their properties, e.g., to increase theirstability in serum and/or to reduce hemolytic activity (Efron, L., etal. (2002) J. Biol. Chem. 277: 24067-24072). In all of these studies,the investigated peptides had antimicrobial activity prior to theattachment of the fatty acids. In addition, it was shown that attachmentof fatty acids to magainin, a well characterized α-helical, positivelycharged antimicrobial peptide, can endow it with antifungal activity(Avrahami, D., et al. (2002) Biochemistry 41: 2254-2263).

Lipopeptides capable of inducing immunological responses, particularlyof cytotoxic T lymphocytes, have been disclosed in U.S. Pat. No.5,871,746. The lipopeptides according to U.S. Pat. No. 5,871,746comprise a peptide having between 10 to 40 amino acids and at least oneantigenic determinant, particularly preferred are peptides derived fromviral proteins.

U.S. Pat. No. 5,837,249 discloses methods for inducing a cytotoxic Tcell response in a mammalian host against a viral infection comprisingadministering to the host a peptide-fatty acid conjugate, the peptidehaving the amino acid sequence corresponding to the amino acid sequenceof a fragment of a glycoprotein or protein of virus.

U.S. Pat. No. 5,583,198 discloses compounds consisting of an amino acidor a peptide linked to a tromethamine derivative or ethanolaminederivative to which one or more fatty acids are optionally linked. Thefatty acids according to U.S. Pat. No. 5,583,198 may enhance theimmunogenic properties of the peptides, enhance their absorption, andprovide slow-release delivery.

Nowhere in the background art is it disclosed or suggested that couplingof fatty acids to positively charged peptides that are inactive orweakly active antibacterial and/or antifungal peptides may impart to theconjugates antibacterial and/or antifungal activity as well as endow theconjugates with selective cytolytic activity against tumor cells.

SUMMARY OF THE INVENTION

The present invention provides safe and effective anti-microbialcompositions. Particularly, the present invention provides compositionsthat can be used against a broad range of microbes including bacteriaand fungi. The compositions of the invention reduce or even eliminatethe need of using antibiotics, and therefore do not promote the growthof antibiotic-resistant bacteria strains as may occur when antibioticsare being used. The present invention further relates to anticancercompositions that do not contain harsh or toxic chemicals. Thecompositions are particularly useful for pharmaceutical applications,especially for topical treatment of microbial infections as well as fortreatment of cancer. The compositions are also useful for hygiene andsanitation, as disinfectants, for food preservation, and foragricultural use.

It is now disclosed that conjugation of a lipophilic moiety,particularly a fatty acid, to an otherwise inactive or weakly activeantimicrobial and/or anticancer peptide can unexpectedly endow thepeptide with superior antimicrobial activity and/or selective cytolyticactivity against tumor cells.

According to one aspect, the present invention provides a lipophilicconjugate comprising a peptide coupled to a fatty acid, the peptidehaving a net positive charge that is equal or greater than +1 comprisingat least two positively charged amino acid residues, said peptide afterconjugation to the fatty acid having at least one activity selected fromthe group consisting of antibacterial, antifungal, and anticanceractivity, wherein the activity after conjugation being higher than priorto conjugation, a cyclic analog, or a salt thereof. In a currentpreferred embodiment, the net positive charge of the peptide is greaterthan +1.

The terms “lipophilic conjugate” and “lipopeptide” used interchangeablythroughout the specification and claims designate a conjugate comprisinga peptide covalently coupled to a fatty acid.

According to one embodiment of the invention, the peptide consists of atleast two amino acid residues. According to currently preferredembodiments, the peptide consists of 3 to 15 amino acid residues. Alipopeptide comprising at least one positively charged amino acid isalso encompassed in the present invention.

It will be understood that the present invention encompasses lipophilicconjugates in which the peptide moiety has overall low hydrophobicity sothat the peptide alone (without the fatty acid) does not significantlyperturb phospholipid membranes and thus does not kill microorganisms.Conjugates comprising the model diastereomeric peptides disclosed inU.S. Pat. No. 6,172,038, WO 98/37090 and in WO 02/40529 are explicitlyincorporated by reference as if fully set forth herein. Thus, thepresent invention relates to known as well as novel peptides that aredevoid or possess very weak antimicrobial and/or anticancer activitywhen not conjugated to a fatty acid in accordance with the principles ofthe invention.

The present invention also provides shorter peptides than thosedisclosed in the background art, inasmuch as the known antimicrobialdiastereomeric peptides disclosed in the art have a minimal length ofsix amino acids. According to the principles of the present inventionthe conjugation of a fatty acid can impart antimicrobial and/oranticancer properties to peptides as short as di- or tri-peptides.

Typically, an inactive antimicrobial peptide is defined as a peptide ofwhich a concentration higher than 100 μM is required to significantlyinhibit bacteria and/or fungi growth. A peptide having low or weakantimicrobial activity is defined as a peptide of which a concentrationbetween 25 to 100 μM is required to significantly inhibit bacteriaand/or fungi growth. Active antimicrobial peptide is defined as apeptide of which a concentration between 10 to 25 μM is required tosignificantly inhibit bacteria and/or fungi growth. Highly activeantimicrobial peptide is defined as a peptide of which a concentrationbetween 5 to 10 μM is required to significantly inhibit bacteria and/orfungi growth, and very highly active antimicrobial peptide is defined asa peptide of which a concentration lower than 5 μM is required tosignificantly inhibit bacteria and/or fungi growth.

According to the present invention, an inactive anticancer peptide isdefined as a peptide of which the LC50 (the concentration at which 50%of the cells die) is higher than 50 μM. A peptide having low or weakanticancer activity is defined as a peptide of which the LC50 is between25 to 50 μM. Active anticancer peptide is defined as a peptide of whichthe LC50 is between 10 to 25 μM. Highly active anticancer peptide isdefined as a peptide of which the LC50 is between 5 to 10 μM, and veryhighly active anticancer peptide is defined as a peptide of which theLC50 is lower than 5 μM. The LC50 is determined in an in-vitro assay.

According to the principles of the present invention, the peptide priorto conjugation of a fatty acid is either inactive or weakly activeantibacterial and/or antifungal and/or anticancer agent. Conjugation ofthe fatty acid endows the peptide with at least one activity selectedfrom antibacterial, antifungal, and anticancer activity so that saidactivity is significantly higher after conjugation than prior toconjugation. Preferably, conjugation of a fatty acid to a peptide of theinvention enhances at least one activity selected from antibacterial,antifungal, and anticancer activity by at least 2 fold, more preferablyby at least 10 fold, and most preferably by at least 20 fold. It will beappreciated that by referring to endowment of a peptide withantibacterial and/or antifungal and/or anticancer activity, this is notintended to imply that the activity affects all bacteria or fungispecies nor all cancer cell types. It is to be understood that theactivity imparted to the previously inactive or weakly active peptideupon conjugation of a fatty acid to said peptide implies that at leastone bacterial species or fungal species or cancer cell type issusceptible to this activity.

The peptides of the present invention comprise L-amino acids, D-aminoacids, or combinations thereof. The amino acids may be selected fromnatural and non-natural amino acids. Peptides having both D-amino acidresidues and L-amino acid residues are defined herein as diastereomericpeptides. Typically, the D-amino acid residues constitute at least onethird of the amino acids of a diastereomeric peptide. The location ofthe D-amino acid residues may vary so long as the antimicrobial and/oranticancer activity of the conjugate is retained. In a currentlypreferred embodiment, the D-amino acid residues are located 1 to 4 aminoacid residues apart.

According to another embodiment of the invention, the fatty acid isselected from the group consisting of saturated, unsaturated,monounsaturated, and polyunsaturated fatty acids. According to currentlypreferred embodiments, the fatty acids consist of at least eight carbonatoms. Examples of the fatty acids that may be coupled to the peptidesof the invention include, but are not limited to, decanoic acid (DA),undecanoic acid (UA), dodecanoic acid (DDA; lauric acid), myristic acid(MA), palmitic acid (PA), stearic acid, arachidic acid, lignoceric acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonicacid, trans-hexadecanoic acid, elaidic acid, lactobacillic acid,tuberculostearic acid, and cerebronic acid. According to currently morepreferred embodiments, the fatty acid is selected from decanoic acid,undecanoic acid, dodecanoic acid, myristic acid, and palmitic acid.

The fatty acid may be coupled to the N-terminal of the peptide, to theC-terminal, or to any other free functional group along the peptidechain, for example, to the c-amino group of lysine. According to certaincurrently preferred embodiments, the fatty acid is coupled to theN-terminus of the peptide.

According to a further embodiment, the lipophilic conjugate comprises afatty acid coupled to a lysine dipeptide, tripeptide, or tetrapeptide.Examples of such conjugates are the lipopeptides of SEQ ID NOS: 1 to 3:

Palmitoyl-Lys-D-Lys-NH₂ SEQ ID No: 1 Palmitoyl-Lys-Lys-Lys-NH₂ SEQ IDNo: 2 Palmitoyl-Lys-D-Lys-Lys-NH₂ SEQ ID No: 3

It should be understood that the fatty acid may be varied and thelipopeptides disclosed are non-limitative exemplary embodiments.

According to another embodiment, the lipophilic conjugate comprises apeptide coupled to a fatty acid, the peptide comprising at least twopositively charged amino acids selected from lysine, arginine,histidine, or a combination thereof, and a hydrophobic amino acidselected from Gly, Ala, Leu, Ile, Val, or a combination thereof.

According to a further embodiment, the lipopeptide comprises at leasttwo Lys residues as the positively charged amino acids and thehydrophobic amino acid is selected from Gly, Ala, Leu, Ile, or Val.Examples of such conjugates are 3-, 4-, 6-9-, 11 and 12-mer lipopeptidesof SEQ ID NOS: 4 to 24:

SEQ ID NO: 4 Palmitoyl-Lys-Gly-Gly-D-Lys-NH₂ SEQ ID NO: 5Palmitoyl-Lys-Leu-D-Leu-Lys-NH₂ SEQ ID NO: 6Palmitoyl-Lys-Ala-D-Ala-Lys-NH₂ SEQ ID NO: 7Palmitoyl-Lys-D-Leu-D-Leu-Leu-Lys-Leu-NH₂ SEQ ID NO: 8Palmitoyl-Lys-D-Ile-D-Ile-Ile-Lys-Ile-NH₂ SEQ ID NO: 9Palmitoyl-Lys-D-Val-D-Val-Val-Lys-Val-NH₂ SEQ ID NO: 10Palmitoyl-Lys-D-Ala-D-Ala-Ala-Lys-Ala-NH₂ SEQ ID NO: 11Palmitoyl-D-Lys-Gly-Gly-Gly-D-Lys-Gly-NH₂ SEQ ID NO: 12Palmitoyl-Lys-Leu-D-Leu-Lys-Leu-Leu-D-Lys-D-Lys- Leu-NH₂ SEQ ID NO: 13:Palmitoyl-Leu-Lys-D-Leu-Leu-Lys-D-Lys-Leu-D-Leu-D- Lys-Lys-Leu-NH₂ SEQID NO: 14: Myristoyl-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-D-Lys-NH₂ SEQ ID NO: 15:Palmitoyl-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-Gly-D- Lys-Gly-Gly-D-Lys-NH₂SEQ ID NO: 16: Palmitoyl-Lys-Ala-D-Ala-D-Ala-Lys-Ala-Ala-D-Ala-Lys-D-Ala-Ala-Lys-NH₂ SEQ ID NO: 17:Palmitoyl-Lys-Val-D-Val-D-Val-Lys-Val-Val-D-Val- Lys-D-Val-Val-Lys-NH₂SEQ ID NO: 18: Palmitoyl-Lys-Ile-D-Ile-D-Ile-Lys-Ile-Ile-D-Ile-Lys-D-Ile-Ile-Lys-NH₂ SEQ ID NO: 19:Undecanoyl-Lys-Leu-D-Leu-D-Leu-Lys-Leu-Leu-D-Leu- Lys-D-Leu-Leu-Lys-NH₂SEQ ID NO: 20: Palmitoyl-Lys-Leu-D-Leu-D-Leu-Lys-Leu-Leu-D-Leu-Lys-D-Leu-Leu-Lys-NH₂ SEQ ID NO: 21:Decanoyl-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ SEQ ID NO: 22:Dodecanoyl-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ SEQ ID NO: 23:Myristoyl-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ SEQ ID NO: 24:Palmitoyl-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂

According to another embodiment, the lipopeptide comprises at least twoarginine or histidine residues as the positively charged amino acids andleucine as the hydrophobic amino acid. Examples of such conjugates arelipopeptides of SEQ ID NO: 25 to 27:

SEQ ID NO: 25: Palmitoyl-Arg-Leu-D-Leu-Arg-NH₂ SEQ ID NO: 26:Dodecanoyl-D-Leu-Arg-Arg-D-Leu-D-Leu-Arg-Arg-D-Leu-D-Leu-Arg-Arg-D-Leu-NH₂ SEQ ID NO: 27:Dodecanoyl-D-Leu-His-His-D-Leu-D-Leu-His-His-D-Leu-D-Leu-His-His-D-Leu-NH₂

According to a further embodiment, the lipopeptide comprises leucine asthe hydrophobic amino acid and a combination of at least two positivelycharged amino acids selected from the group consisting of lysine,arginine, and histidine. Examples of such conjugates are lipopeptides ofSEQ ID NOS: 28 to 33:

SEQ ID NO: 28: Palmitoyl-Lys-Leu-D-Leu-Arg-Leu-Leu-D-Lys-D-Lys-Leu-D-Leu-Arg-NH₂ SEQ ID NO: 29:Palmitoyl-Lys-Leu-D-Leu-Leu-Arg-D-Leu-Leu-D-Lys-D- Lys-Leu-Leu-Arg-NH₂SEQ ID NO: 30: Palmitoyl-Lys-Leu-D-Leu-Arg-Leu-Leu-D-Lys-D-Lys-Leu-D-Leu-Arg-Leu-NH₂ SEQ ID NO: 31:Palmitoyl-Lys-Leu-D-Leu-Leu-Arg-D-Leu-Leu-D-Lys-D-Lys-Leu-Leu-Arg-D-Leu-Lys-NH₂ SEQ ID NO: 32:Dodecanoyl-D-Leu-Arg-His-D-Leu-D-Leu-Arg-His-D-Leu-D-Leu-Arg-His-D-Leu-NH₂ SEQ ID NO: 33:Dodecanoyl-D-Leu-Lys-His-D-Leu-D-Leu-Lys-His-D-Leu-D-Leu-Lys-His-D-Leu-NH₂

According to another embodiment, the lipopeptide comprises at least twopositively charged amino acids and a combination of hydrophobic and/ornon-hydrophobic amino acids. In one particular embodiment, thehydrophobic and non-hydrophobic amino acids are selected from Leu, Gly,Ala, Ser, Thr, and Met, and examples thereof are the 7-mer lipopeptidesof SEQ ID NOS: 34 to 35:

SEQ ID NO: 34 Palmitoyl-Leu-D-Leu-Leu-Arg-D-Leu-Gly-Leu-NH₂ SEQ ID NO:35 Palmitoyl-Leu-D-Leu-Lys-Leu-Leu-D-Lys-Gly-NH₂

According to a further embodiment, the conjugate comprises a peptidecomprising at least two positively charged amino acids and a negativelycharged amino acid. In a currently preferred embodiment, the positivelycharged amino acid is lysine. Examples of such conjugates are the S— and4-mer lipopeptides of SEQ ID NOS: 36 to 38:

Palmitoyl-Glu-Lys-D-Lys-Lys-NH₂ SEQ ID NO: 36Palmitoyl-Lys-D-Lys-Glu-Lys-NH₂ SEQ ID NO: 37Palmitoyl-Glu-D-Lys-Lys-NH₂ SEQ ID NO: 38

According to still a further embodiment, the invention relates to cyclicanalogs of the lipophilic conjugates. Examples of such cyclic conjugatesare the lipopeptides of SEQ ID NO: 39 to 46:

According to another aspect, the present invention provides apharmaceutical composition comprising as an active ingredient alipophilic conjugate comprising a peptide coupled to a fatty acid, thepeptide having a net positive charge that is equal or greater than +1comprising at least two positively charged amino acid residues, saidpeptide after conjugation to the fatty acid having at least one activityselected from the group consisting of antibacterial, antifungal, andanticancer activity, wherein the activity after conjugation being higherthan prior to conjugation, a cyclic analog, or a salt thereof.

The pharmaceutical composition may be formulated for any route ofadministration including, but not limited to, intravenous,intramuscular, intraperitoneal, nasal, oral, intralesional and topical.In currently preferred embodiments, the pharmaceutical composition isformulated for topical or intralesional administration.

According to a further aspect, the present invention provides acomposition comprising as an active ingredient a lipophilic conjugate ofthe invention useful for hygienic purposes, as a disinfectant, for foodpreservation, for veterinary use, or for agricultural use.

According to a further aspect, the present invention provides a methodfor treating an infection caused by pathogenic organisms in a subjectcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising a lipophilicconjugate of the invention and a pharmaceutically acceptable carrier.

Infections that may be treated by the pharmaceutical composition of theinvention include, but are not limited to, topical infections caused bypathogenic organisms such as bacterial infections, particularlyinfections caused by bacteria resistant to antibiotics, and infectionscaused by pathogenic fungi.

According to a further aspect, the present invention provides a methodfor treating cancer in a subject comprising administering to a subjectin need thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a lipophilic conjugate of the invention and apharmaceutically acceptable carrier.

According to one embodiment, cancers that may be treated by thepharmaceutical compositions of the invention include benign andmalignant solid or non-solid tumors.

In a further aspect, the present invention provides a method fordisinfecting an object comprising contacting an object with amicrobicidally effective amount of a disinfecting composition comprisingas an active ingredient a lipophilic conjugate of the invention.

These and other embodiments of the present invention will be betterunderstood in relation to the description, figures, examples, and claimsthat follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the lack of hemolytic activity of short lipopeptides having1 to 3 amino acid residues at concentrations lower than 25 μM,concentrations at which the lipopeptides exert antimicrobial activity.Human red blood cells were incubated in the presence of increasingconcentrations of the lipopeptides for 1 h, and the release ofhemoglobin was monitored by measuring the absorbance at 540 nm.

FIG. 2 shows the lack of hemolytic activity of short lipopeptides having4 amino acid residues at concentrations lower than 25 μM, concentrationsat which the lipopeptides exert antimicrobial activity. The hemolyticactivity was monitored by measuring the absorbance at 540 nm.

FIG. 3 shows the lack of hemolytic activity of additional shortlipopeptides having 4 amino acid residues at concentrations lower than12.5 μM, concentrations at which the lipopeptides exert antimicrobialactivity. The hemolytic activity was monitored by measuring theabsorbance at 540 nm.

FIG. 4 shows the lack of hemolytic activity of short lipopeptides having2 to 4 amino acid residues at concentrations lower than 12.5 μM,concentrations at which the lipopeptides exert antimicrobial activity.The hemolytic activity was monitored by measuring the absorbance at 540nm.

FIG. 5 shows the hemolytic activity of lipopeptides having 12 amino acidresidues. Note that at concentrations lower than 12.5 μM, lipopeptides2, 4, and 5 were not hemolytic, and at concentrations lower than 5 μM,peptides 6 to 8 were not hemolytic. The hemolytic activity was monitoredby measuring the absorbance at 540 nm.

FIG. 6 shows the effect of different fatty acids on the hemolyticactivity of lipopeptides. A 12-mer diastereomeric peptide was coupledeither to decanoic acid, dodecanoic acid, myristic acid or to palmiticacid and the hemolytic activity of the conjugates was monitored bymeasuring the absorbance at 540 nm.

FIGS. 7A-B show the hemolytic activity of lipopeptides having the samefatty acid, but varying in the peptide moiety. Dodecanoic acid wascoupled to 12-mer peptides, which are composed of six Leu residues andsix positively charged amino acid residues of either Lys, His, Arg, or acombination thereof. The hemolytic activity of the lipopeptides wasdetermined at neutral pH (FIG. 7A) or at acidic pH (FIG. 7B) bymeasuring the absorbance at 540 nm.

DETAILED DESCRIPTION OF THE INVENTION

Diastereomeric peptides, which have been shown to be active againstbacteria, have a narrow spectrum of activity against fungi, especiallyagainst filamentous fungi. In order to improve the cytolytic activity ofL-amino acid peptides or of diastereomeric peptides against fungi, or tomodify an inactive peptide such that it will be active against fungiand/or bacteria and/or tumor cell, fatty acids (having variable lengths)were attached to the peptides at either the N-terminus, the C-terminus,or along the peptide chain. Several parameters such as the length of thepeptide, the location and number of D-amino acids, the polarity of thediastereomeric peptide, and the length of the fatty acid were found toaffect the peptide potency, selectivity and spectrum of activity towardspathogenic cells such as bacteria, fungi, and cancer cells.

Due to the increased structural and sequence flexibility of thelipopeptides of the invention, this family of lipopeptides provides anefficient alternative to the known native amphipathic α-helicalantimicrobial peptides having a complex sequence of hydrophobic andpolar amino acid residues, and hence provides important advantages forthe design of a repertoire of potent anti-pathogenic lipopeptides forthe treatment of diseases.

The present invention provides lipophilic conjugates comprising apeptide coupled to a fatty acid, the peptide having a net positivecharge that is equal or greater than +1 comprising at least twopositively charged amino acid residues, said peptide after conjugationto the fatty acid having at least one activity selected from the groupconsisting of antibacterial, antifungal, and anticancer activity,wherein the activity after conjugation being higher than prior toconjugation, a cyclic analog, or a salt thereof. Preferably, the netpositive charge of the peptide is greater than +1.

The term “diastereomeric peptide” as used herein refers to a peptidecomprising both L-amino acid residues and D-amino acid residues. Theamino acid residues are represented throughout the specification andclaims by three-letter codes according to IUPAC conventions. When thereis no indication, the amino acid residue occurs in L isomerconfiguration. Amino acid residues present in D isomer configuration areindicated by “D” before the residue abbreviation.

The term “antimicrobial activity” as used herein refers to lyticactivity against microorganisms. Particularly, the antimicrobialactivity refers to antifungal activity and/or antibacterial activity.However, activity against other pathogenic organisms such as viruses,mycoplasma, and protozoa is also contemplated in the present invention.

The term “anticancer activity” as used herein refers to preferentialcytotoxic effect against tumor cells without any significant adverseeffects to normal cells under the same conditions of exposure. It willbe understood that the enhanced cytotoxic effect of the lipophilicconjugates of the invention against tumor cells compared to normal cellsdepends primarily upon the metabolic activity of the cells. Thus, whiletumor cells proliferate at high rates and hence have increased metabolicactivity, these cells are more affected by the lipophilic conjugates ofthe invention, whereas normal cells, which typically exhibit lowermetabolic activity compared to tumor cells, are less affected by thelipophilic conjugates of the invention. The cytotoxic effect under invitro or in vivo conditions is detected by various means known in theart, for example, by measuring thymidine incorporation into cells, bymetabolic assays using MTT, XTT, or AlamarBlue fluorescent reagents, andby gadolinium enhanced MRI scanning.

Typically, an inactive antimicrobial peptide is defined as a peptide ofwhich a concentration higher than 100 μM is required to significantlyinhibit bacteria and/or fungi growth. A peptide having low antimicrobialactivity is defined as a peptide of which a concentration between 25 to100 μM is required to significantly inhibit bacteria and/or fungigrowth. Active peptide is defined as a peptide of which a concentrationbetween 10 to 25 μM is required to significantly inhibit bacteria and/orfungi growth. Highly active peptide is defined as a peptide of which aconcentration between 5 to 10 μM is required to significantly inhibitbacteria and/or fungi growth, and very highly active peptide is definedas a peptide of which a concentration lower than 5 μM is required tosignificantly inhibit bacteria and/or fungi growth.

Typically, an inactive anticancer peptide is defined as a peptide ofwhich the LC50 (the concentration at which 50% of the cells die) ishigher than 50 μM. A peptide having low or weak anticancer activity isdefined as a peptide of which the LC50 is between 25 to 50 μM. Activeanticancer peptide is defined as a peptide of which the LC50 is between10 to 25 μM. Highly active anticancer peptide is defined as a peptide ofwhich the LC50 is between 5 to 10 μM, and very highly active anticancerpeptide is defined as a peptide of which the LC50 is lower than 5 μM.The LC50 is determined in an in-vitro assay.

The terms “lipopeptide” and “lipophilic conjugate” as used herein referto a peptide covalently coupled to a fatty acid. The terms lipopeptideand lipophilic conjugate are used interchangeably throughout thespecification and claims.

The lipopeptide of the invention comprises at least two amino acidresidues. In currently preferred embodiments, the lipopeptides comprise3-15 amino acid residues. However, a lipopeptide comprising at least onepositively charged amino acid is also contemplated in the invention. Itshould be understood that lipopeptides in which the peptide moiety hasoverall low hydrophobicity that does not allow the peptide alone(without the fatty acid) to perturb phospholipid membranes and to killmicroorganisms are contemplated in the invention.

The peptides of the present invention can be synthesized using methodswell known in the art including chemical synthesis and recombinant DNAtechnology. Synthesis may be performed by solid phase peptide synthesisdescribed by Merrifield (see J. Am. Chem. Soc., 85:2149, 1964).Alternatively, the peptides of the present invention can be synthesizedusing standard solution methods (see, for example, Bodanszky, M.,Principles of Peptide Synthesis, Springer-Verlag, 1984). Preferably, thepeptides of the invention are synthesized by solid phase peptidesynthesis as exemplified herein below (Example 1). Cyclization of thelipopeptides of the invention can be performed by methods known in theart, for example, by inserting two cysteine residues or analogs thereofto form a disulfide bond (see Examples herein below and Unger et al.(2001) Biochemistry 40: 6388-6397). Cyclization may also be performedbetween the carboxyl and amino termini of the peptide. Alternatively oradditionally, cyclization may be performed between a functional group ofan amino acid, for example an ε-amino group of Lys, and the carboxylterminus of the peptide (see Examples herein below and Tsubery et al.,(2000) J. Med. Chem. 43: 3085-3092). Thus, the present inventionencompasses any cyclic analog of linear peptides disclosed in thepresent invention.

The invention contemplates lipophilic conjugates comprising peptidescomposed of natural amino acids, non-natural amino acids, and analogsthereof. Examples of non-natural amino acids are norleucine, ornithine,citrulline, diaminobutyric acid, homoserine, isopropyl Lys,3-(2′-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and3-(3′-pyridyl-Ala). The net positive charge of a peptide of theinvention is due to the amino acid composition of the peptide, butderivatization of non-charged amino acid residues to yield positivelycharged amino acids as known in the art, for example by methylation, iscontemplated in the present invention.

Positively charged amino acids as used herein are selected frompositively charged amino acids known in the art. Examples of positivelycharged amino acids are lysine, arginine, and histidine. Hydrophobicamino acids as used herein are selected from hydrophobic amino acidsknown in the art. Examples of hydrophobic amino acids are leucine,isoleucine, glycine, alanine, and valine. Negatively charged amino acidsare selected from negatively charged amino acids known in the artincluding, but not limited to, glutamic acid and aspartic acid.

The fatty acid that can be coupled to the peptides of the invention isselected from saturated, unsaturated, monounsaturated, andpolyunsaturated fatty acids. Typically, the fatty acid consists of atleast eight carbon atoms, such as, for example, decanoic acid (DA),undecanoic acid (UA), dodecanoic acid (lauric acid), myristic acid (MA),palmitic acid (PA), stearic acid, arachidic acid, lignoceric acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonicacid, trans-hexadecanoic acid, elaidic acid, lactobacillic acid,tuberculostearic acid, and cerebronic acid. The fatty acid may becoupled to the N-terminal, to the C-terminal, or to any other freefunctional group along the peptide chain, for example, to the ε-aminogroup of lysine. Coupling of a fatty acid to a peptide is performedsimilarly to the coupling of an amino acid to a peptide during peptidesynthesis (Examples herein below). It should be understood that thefatty acid is covalently coupled to the peptide. The terms “coupling”and “conjugation” are used herein interchangeably and refer to thechemical reaction, which results in covalent attachment of a fatty acidto a peptide to yield a lipophilic conjugate.

According to some embodiments of the invention, a short peptide (2- to4-mer peptide) is coupled to a long aliphatic chain fatty acid while along peptide, e.g., 12-mer peptide, is coupled to a short aliphaticchain fatty acid. Thus, for example, a peptide consisting of from two tofour amino acid residues and having little or no antimicrobial activityat concentrations below 25 μM is coupled to a fatty acid that consistsof at least 16 carbon atoms such as, for example, palmitic acid. Also, apeptide consisting of at least five amino acid residues is coupled to afatty acid having at least 10 carbon atoms such as, for example,undecanoic acid or myristic acid. However, it should be understood thatany fatty acid having at least eight carbon atoms could be coupled tothe peptides of the invention so long as the antimicrobial and/oranticancer activity of the conjugate is enhanced. Though we do not wishto be bound to any mechanism of action, it will be appreciated thatcoupling of a fatty acid to a peptide is aimed at increasing peptidehydrophobicity, optionally its oligomerization in solution, and thusendowing it with antimicrobial and/or anticancer activity.

The present invention provides pharmaceutical compositions comprisingthe lipophilic conjugates of the invention and a cosmetically and/orpharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to a vehicle which delivers the activecomponents to the intended target and which does not cause harm tohumans or other recipient organisms. As used herein, “pharmaceutical”will be understood to encompass both human and animal pharmaceuticals.Useful carriers include, for example, water, acetone, ethanol, ethyleneglycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, or mineral oil. Methodology and components forformulation of pharmaceutical compositions are well known, and can befound, for example, in Remington's Pharmaceutical Sciences, EighteenthEdition, A. R. Gennaro, Ed., Mack Publishing Co. Easton Pa., 1990. Thepharmaceutical composition may be formulated in any form appropriate tothe mode of administration, for example, solutions, colloidaldispersions, emulsions (oil-in-water or water-in-oil), suspensions,creams, lotions, gels, foams, sprays, aerosol, ointment, tablets,suppositories, and the like.

The pharmaceutical compositions can also comprise other optionalmaterials, which may be chosen depending on the carrier and/or theintended use of the composition. Additional components include, but arenot limited to, antioxidants, chelating agents, emulsion stabilizers,e.g., carbomer, preservatives, e.g., methyl paraben, fragrances,humectants, e.g., glycerin, waterproofing agents, e.g., PVP/EicoseneCopolymer, water soluble film-formers, e.g., hydroxypropylmethylcellulose, oil-soluble film formers, cationic or anionic polymers,and the like.

The pharmaceutical compositions useful in the practice of the presentinvention comprise a lipopeptide of the invention optionally formulatedinto the pharmaceutical composition as a pharmaceutically acceptablesalt form. Pharmaceutically acceptable salts include the acid additionsalts (formed with the free amino groups of the polypeptide), which areformed with inorganic acids, such as for example, hydrochloric orphosphoric acid, or with organic acids such as acetic, oxalic, tartaric,and the like.

Suitable bases capable of forming salts with the lipopeptides of thepresent invention include, but are not limited to, inorganic bases suchas sodium hydroxide, ammonium hydroxide, potassium hydroxide and thelike; and organic bases such as mono-, di- and tri-alkyl and aryl amines(e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl amine andthe like) and optionally substituted ethanolamines (e.g. ethanolamine,diethanolamine and the like).

The lipophilic conjugates of the invention can be used individually orin combination with other components for disinfecting objects. The term“disinfecting” relates to preventing, inhibiting, and/or alleviatingmicrobial growth, and is used interchangeably with sterilizing. Theamount of each component used will depend on the purpose of the use,e.g., disinfecting medical or surgical equipment, and disinfectingtissue culture equipment, media, incubators, hoods, dishes, and thelike. The compositions may also be used for treating contact lenses,such as disinfecting solutions, cleaning products and products forenhancing the ocular comfort of patients wearing contact lenses; othertypes of ophthalmic compositions, such as ocular lubricating products,artificial tears, and the like. The concentration determined to benecessary for the above-stated purposes can be functionally described as“an amount effective to disinfect” or “microbicidally effective amount”or variations thereof.

The lipopeptides of the invention may also be used for foodpreservation, in veterinary compositions as alternative to antibiotics,or for agricultural use.

The present invention provides methods for treating an infection in asubject comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a lipophilicconjugate of the invention and a pharmaceutically acceptable carrier.

The present invention also provides methods for treating cancer in asubject comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a lipophilicconjugate of the invention and a pharmaceutically acceptable carrier.

As used herein, the term “treating” means remedial treatment, andencompasses the terms “reducing”, “suppressing”, “ameliorating” and“inhibiting”, which have their commonly understood meaning of decreasingor arresting an infection and/or decreasing or arresting tumor growth.

The term “therapeutically effective amount” as used herein refers to anamount of the pharmaceutical composition that when administered to asubject is capable of exerting antifungal and/or antibacterial and/oranticancer activity. Assays for detecting the antifungal and/orantibacterial and/or anticancer activity are well known in the art andinclude, but are not limited to, in vitro assays for bacterial growth,fungal growth, and tumor cell growth as described herein below (Examples3, 4, and 7 herein below). Thus, according to the present invention, atherapeutically effective amount is an amount that when administered toa subject is sufficient to inhibit, preferably to eradicate, bacterialand/or fungal infection and/or cancer growth. According to the presentinvention, a subject is an animal, preferably a mammal, and morepreferably a human.

According to the present invention, the step of administering thepharmaceutical compositions of the invention includes any route ofadministration including, but not limited to, intravenous,intraarterial, intramuscular, intraperitoneal, oral, ophthalmic, nasal,vaginal, rectal, intralesional, and topical. Particularly, thepharmaceutical compositions of the invention are useful for topical andintralesional application. As used herein, the term “topical” means“pertaining to a particular surface area” and the topical agent appliedto a certain area of said surface will affect only the area to which itis applied. Thus, any and all applications in which the lipophilicconjugates act locally and not through the blood circulation are alsoencompassed in the present invention. It should be also appreciated thatlocal administration of the pharmaceutical compositions of the inventiondirectly into a tumor or adjacent to the tumor are highly useful.

The lipopeptides of the invention may be used in topical applicationsagainst a wide variety of topical infections. Such applications include,but are not limited to, treatment of bacterial and fungal infectionsincluding treatment of acne, fungal infections of the scalp, fungal orbacterial infections related to surgical or traumatic wounds, chronic orpoorly healing skin lesions (especially in diabetes), vaginal infections(vaginitis), eye and ear infections, burn wounds, infections of mouthand throat, localized infections such as chronic pulmonary infections incystic fibrosis, emphysema and asthma.

Topical infections are characterized by opportunistic colonization of awide range of endogenous and exogenous pathogenic cells. Treatment ofsevere wounds such as burns or poorly healing wounds, e.g., foot ulcersin diabetes mellitus patients, require long-term administration ofantibiotics, which leads to selection of resistant bacteria such asStreptococcus pyogenes or the methicilin-resistant Staphylococcusaureus. These problems could be overcome by the lipopeptides of theinvention due to their wide spectrum of activity and their ability toact against non-resistant and resistant bacteria and fungi. The observedresistance of the lipopeptides to proteolytic digestion may enable themto reach the digestive system in intact form and to eliminate therebacterial infections such as chronic gastric mucosal infestation byHelicobacter pylori and intestinal bacterial infections.

The activity of the lipopeptides against different strains of fungiindicate their potential use for the treatment of nail fungus such as:(i) onychomycoses, the most current nail infection caused mainly bydermatophytes, in particular by Trichophyton rubrum, and less frequentlyby Trichophyton mentagrophytes and Epidermophyton floccosum; (ii)infections caused by mold; and (iii) infections caused by yeasts,particularly Candida albicans, as in chronic paronychia and onycholysis,and chronic mucocutaneous candidosis.

The pharmaceutical compositions can be used in the treatment of benignor malignant solid or non-solid tumors. As the lipopeptides of theinvention inhibit tumor cell growth (see Example 7 herein below), theuse of these conjugates can be highly advantageous in treating cancerdiseases.

All types of cancers may be included in the scope of the presentinvention. As a non limiting example, the following cancers can betreated: skin (e.g., squamous cell carcinoma, basal cell carcinoma, ormelanoma), breast, colorectal, prostate, brain and nervous system, headand neck, testicular, ovarian, pancreatic, lung, liver (e.g., hepatoma),kidney, bladder, gastrointestinal, bone, endocrine system (e.g., thyroidand pituitary tumors), and lymphatic system (e.g., Hodgkin's andnon-Hodgkin's lymphomas) cancers. Cancers of the nervous system include,for example, astrocytoma, pligodendroglioma, menigioma, neuroblastoma,glioblastoma, ependyoma, Schwannoma, neurofibrosarcoma, neuroblastoma,and medullablastoma. Other types of cancer include fibrosarcoma,epidermoid carcinoma, and any other cancer that form solid tumors. Alsocontemplated in the present invention benign proliferative diseases ofthe blood and malignant proliferative diseases of the blood, forexample, leukemia.

Additionally, the pharmaceutical compositions of the present inventionare well suited for combination with other active components intendedfor topical, intralesional, or any other type of application. Forexample, the methods of treating cancer in a subject can be carried outin conjunction with chemotherapy or radiotherapy. Thus, thepharmaceutical compositions of the invention may be administered withchemotherapeutic agents. Examples of chemotherapeutic agents that may beused include alkylating agents, antineoplastic antibiotics,antimetabolites, and a like.

It is further understood that the amount of the pharmaceuticalcomposition administered to any particular subject will depend upon avariety of factors including, but not limited to, the type, location,and extent of the microbial infection, the extent, density, location,and type of tumor cells to be killed as well as the age, body weight,general health, and gender of the subject, and the route ofadministration. Administration of the pharmaceutical composition shouldbe continued until the infection eradicated or the tumor regresses andhealth has been restored to the subject.

The present invention further provides methods for disinfecting anobject comprising contacting an object with a microbicidally effectiveamount of a disinfecting composition comprising a lipophilic conjugateof the invention.

EXAMPLES

The invention will now be described with reference to some non-limitingexamples.

Experimental Procedures (i) Materials

4-Methyl benzhydrylamine resin (BHA) and butyloxycarbonyl (Boc) aminoacids were purchased from Calbiochem-Novabiochem Co. (La Jolla, Calif.,USA). Other reagents used for peptide synthesis included trifluoroaceticacid (TFA, Sigma), N,N-diisopropylethylamine (DIEA, Sigma),dicyclohexylcarbodiimide (DCC, Fluka), 1-hydroxybenzotriazole (1-HOBT,Pierce), and dimethylformamide (DMF, peptide synthesis grade, Biolab,IL). All other reagents were of analytical grade. Buffers were preparedin double-distilled water.

(ii) Peptide Synthesis, Acylation and Purification

Peptides were synthesized by a solid phase method on 4-methylbenzhydrylamine resin (BHA) (0.05 meq) (Merrifield et. al., 1982; Shaiet. al., 1990). The resin-bound peptides were cleaved from the resin byhydrogen fluoride (HF) and, after HF evaporation, and washing with dryether, extracted with 50% acetonitrile/water. HF cleavage of thepeptides bound to BHA resin resulted in C-terminus amidated peptides.Each crude peptide contained one major peak, as revealed by RP-HPLC(reverse phase high-performance liquid chromatography) that was 60-80%pure peptide by weight. The synthesized peptides were further purifiedby RP-HPLC on a C₁₈ reverse phase Bio-Rad semi-preparative column(250×10 mm, 300 nm pore size, 5-μm particle size). The column was elutedin 40 min, using a linear gradient of 25-60% acetonitrile in water, bothcontaining 0.05% TFA (v/v), at a flow rate of 1.8 ml/min. The purifiedpeptides, which were shown to be homogeneous (˜95%) by analytical HPLC,were subjected to amino acid analysis and electrospray mass spectroscopyto confirm their composition and molecular weight. The fatty acid wasconjugated to the N-terminus of the peptides using the same protocolused to attach protected amino acids for peptide synthesis.

(iii) Synthesis of the Cyclic Lipopeptides.

The cyclic peptides were synthesized by a solid-phase method asdescribed in section (ii) above, without or with cysteine residues atboth the N- and C-termini of the peptides. The cyclization withoutcysteine is carried out by protecting the N-terminal, activating theC-terminal, deprotecting the N-terminal, and reacting the C- andN-terminal groups while still bound to the resin. The fatty acids, inthis specific case, are attached to one of the lysines. When the peptidecontains cysteine residues at both the N- and C-termini, after acylationof the N-terminal or another lysine in the backbone, HF cleavage andRP-HPLC purification, the peptides are solubilized at low concentrationin PBS (pH 7.3), and cyclization is completed after 12 h. The cyclicpeptides are further purified on RP-HPLC and subjected to amino acidanalysis to confirm their composition, and SDS-PAGE to confirm theirmonomeric state.

Example 1 Synthesis of 12-Mer Linear Lipopeptides and Cyclic Peptides

The following lipopeptides were synthesized as described in theExperimental Procedures sections (ii) and (iii). Decanoic acid (DA),undecanoic (UA), dodecanoic acid (DDA), myristic (MA), or palmitic (PA)acid were conjugated to 12-mer C-amidated all L-amino acid ordiastereomeric peptides to yield the lipophilic conjugates. The peptidescontained a hydrophobic amino acid selected from Gly, Ala, Val, or Leuand the positively charged amino acid Lys. Each of the diastereomericpeptides contained 4-6 amino acid residues in the D-configuration. Thelipopeptides will be designated hereinafter by numerals.

1 [D]-K^(1,5,9,12)-K₄G₈-MA of the sequence:(CH₃—(CH₂)₁₂—CO—)-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-D-Lys-NH₂2 [D]-K^(1,5,9,12)-K₄G₈-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-Gly-D-Lys-Gly-Gly-D-Lys-NH₂3 K₄G₈-MA of the sequence:(CH₃—(CH₂)₁₂—CO—)-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Lys-NH₂4 K₄G₈-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Gly-Lys-Gly-Gly-Lys-NH₂5 [D]-A^(3,4,8,10)-K₄A₈-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-Lys-Ala-D-Ala-D-Ala-Lys-Ala-Ala-D-Ala-Lys-D-Ala-Ala-Lys-NH₂6 K₄A₈-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-Lys-Ala-Ala-Ala-Lys-Ala-Ala-Ala-Lys-Ala-Ala-Lys-NH₂7 [D]-V^(3,4,8,10)-K₄V₈-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-Lys-Val-D-Val-D-Val-Lys-Val-Val-D-Val-Lys-D-Val-Val-Lys-NH₂8[D]-L^(3,4,8,10)-K₄L₈-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-Lys-Leu-D-Leu-D-Leu-Lys-Leu-Leu-D-Leu-Lys-D-Leu-Leu-Lys-NH₂9 [D]-L^(3,4,8,10)-K₄L₈-MA of the sequence:(CH₃—(CH₂)₁₂—CO—)-Lys-Leu-D-Leu-D-Leu-Lys-Leu-Leu-D-Leu-Lys-D-Leu-Leu-Lys-NH₂10 [D]-L^(3,4,8,10)-K₄L₈-UA of the sequence:(CH₃—(CH₂)₉—CO—)-Lys-Leu-D-Leu-D-Leu-Lys-Leu-Leu-D-Leu-Lys-D-Leu-Leu-Lys-NH₂11 [D]-L^(1,4,5,8,9,12)-L₆K₆-DA of the sequence:(CH₃—(CH₂)₈—CO—)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂12 [D]-L^(1,4,5,8,9,12)-L₆K₆-DDA of the sequence:(CH₃—(CH₂)₁₀—CO—)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂13 [D]-L^(1,4,5,8,9,12)-L₆K₆-MA of the sequence:(CH₃—(CH₂)₁₂—CO—)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂14 [D]-L^(1,4,5,8,9,12)-L₆K₆-PA of the sequence:(CH₃—(CH₂)₁₄—CO—)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂

The following cyclic amidated lipopeptides 15 to 20 were prepared:

Additional linear lipopeptides were synthesized, the peptides containedLeu as the hydrophobic amino acid residue and Arg, His, Lys, or acombination thereof as the positively charged amino acid residues:

21 [D]-L^(1,4,5,8,9,12)-L₆K₆-DA of the sequence:(CH₃-(CH₂)₈-CO-)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ 22 [D]-L^(1,4,5,8,9,12)-L₆K₆-DDA ofthe sequence: (CH₃-(CH₂)₁₀-CO-)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ 23 [D]-L^(1,4,5,8,9,12)-L₆K₆-MA of thesequence: (CH₃-(CH₂)₁₂-CO-)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ 24 [D]-L^(1,4,5,8,9,12)-L₆K₆-PA of thesequence: (CH₃-(CH₂)₁₄-CO-)-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-D-Leu-Lys-Lys-D-Leu-NH₂ 25 [D]-L^(1,4,5,8,9,12)-L₆R₆-DDA ofthe sequence: (CH₃-(CH₂)₁₀-CO-)-D-Leu-Arg-Arg-D-Leu-D-Leu-Arg-Arg-D-Leu-D-Leu-Arg-Arg-D-Leu-NH₂ 26 [D]-L^(1,4,5,8,9,12)-L₆H₆-DDA ofthe sequence: (CH₃-(CH₂)₁₀-CO-)-D-Leu-His-His-D-Leu-D-Leu-His-His-D-Leu-D-Leu-His-His-D-Leu-NH₂ 27 [D]-L^(1,4,5,8,9,12)-L₆R₃H₃-DDA ofthe sequence: (CH₃-(CH₂)₁₀-CO-)-D-Leu-Arg-His-D-Leu-D-Leu-Arg-His-D-Leu-D-Leu-Arg-His-D-Leu-NH₂ 28 [D]-L^(1,4,5,8,9,12)-L₆K₃H₃-DDA ofthe sequence: (CH₃-(CH₂)₁₀-CO-)-D-Leu-Lys-His-D-Leu-D-Leu-Lys-His-D-Leu-D-Leu-Lys-His-D-Leu-NH₂

Example 2 Mass Spectrometry and Retention Time of the Lipopeptides

The lipopeptides were subjected to C₄ RP-HPLC to determine the effect ofthe fatty acid on the retention time of the lipophilic conjugates.

The amidated lipopeptides were applied on an analytical C₄ column andeluted within 80 min using a linear gradient of 0 to 80% acetonitrile inwater containing 0.05% TFA (v/v). The molecular weight of thelipopeptides was obtained by mass spectrometry.

TABLE 1 Sequence, molecular weight, and retention time of thelipopeptides. Calculated RP-HPLC Molecular Retention Conjugate WeightTime Designation Sequence (gr/mole) (minutes) [D]-L₆K₆ L KK L L K KL L K 1466 16.3 K L-NH₂ [D]-L₆K₆- CH₃(CH₂)₈CO-L KK L 1620.3 29.9 DA-21L K K L L K K L- NH₂ [D]-L₆K₆- CH₃(CH₂)₁₀CO-L KK 1648.3 32.9 DDA-22L L K K L L K K L- NH₂ [D]-L₆K₆- CH₃(CH₂)₁₂CO-L KK 1676.3 36.1 MA-23L L K K L L K K L- NH₂ [D]-L₆K₆- CH₃(CH₂)₁₄CO-L KK 1704.3 41.5 PA-24L L K K L L K K L- NH₂Note: The D-amino acid residues are denoted by bold letters andunderline.

Table 1 shows that as the length of the aliphatic chain of the fattyacid increases, the retention time as well as the molecular weight ofthe lipophilic conjugate increase.

TABLE 2 Sequence and retention time of the lipopeptides. ConjugateRP-HPLC Retention Designation Sequence Time (minutes) DL6R6 LRRLLRRLLRRL15.5 DDA-DL6R6-25 DDA-LRRLLRRLLRRL 25.7 DL6H6 LHHLLHHLLHHL 16DDA-DL6H6-26 DDA-LHHLLHHLLHHL 28 DL6R3H3 LRHLLRHLLRHL 18.6DDA-DL6R3H3-27 DDA-LRHLLRHLLRHL 27 DL6K6 LKKLLKKLLKKL 14.3 DDA-DL6K6-22DDA-LKKLLKKLLKKL 24.3 DL6K3H3 LKHLLKHLLKHL 15.5 DDA-DL6K3H3-28DDA-LKHLLKHLLKHL 26

Table 2 shows that as the length of the aliphatic chain of the fattyacid increases, the retention time of the lipophilic conjugateincreases.

Example 3 Antibacterial Activity of the Lipopeptides at pH 7.4 and pH5.5

The antibacterial activity of the lipopeptides was examined in sterile96-well plates (Nunc F96 microtiter plates) in a final volume of 100 μlas follows: Aliquots (50 μl) of a suspension containing bacteria atconcentration of 1×10⁶ Colony-Forming Units (CFU)/ml in culture LB(Lauria broth) medium (pH 7.4 or pH 5.5) were added to 50 μl of watercontaining the lipopeptide in serial 2-fold dilutions in water.Adjustment of the pH was done by diluted solutions of HCl and NaOHInhibition of growth was determined by measuring the absorbance at 600nm with a Microplate autoreader E1309 (Bio-Tek Instruments), after anincubation time of 18-20 h at 37° C. Antibacterial activities wereexpressed as the minimal inhibitory concentration (MIC), theconcentration at which 100% inhibition of growth was observed after18-20 h of incubation. The bacteria used were: Escherichia coli ATCC25922, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC 6538P, Enterococcus faecalis ATCC29212, Enterobacter cloacae ATCC 49141. The antibacterial activity oflipopeptides was also examined against resistant bacteria:methicilin-resistant Staphylococcus aureus (MRSA) ATCC 700698 andvancomycin-resistant Enterococcus faecium (VRE) ATCC 700221. If notindicated otherwise, the bacterial growth was detected at pH 7.4.

TABLE 3 Minimal Inhibitory Concentration (μM) of short lipopeptides onbacteria growth. S. aureus A. baumannii E. coli- P. aeruginosa ConjugateATCC 6538P ATCC 19606 D21 ATCC 27853 M. laleus PA-KKKK 25 nd 12.5 3.123.12 PA-KGGK 50 nd 25 12.5 3.12 PA-KLLK 3.12 nd 100 100 3.12 PA-KAAK6.25 100 6.25 100 nd PA-K >100 >100 >100 >100 nd PA-KK 6.25 100 6.25 1003.12 PA-KKK 12.5 12.5 6.25 50 3.12 PA-KKK 6.25 12.5 6.25 25 nd MYR-KKK12.5 >100 25 nd PA-EKKK 12.5 >100 25 >100 nd PA-RLLR 25 >100 >100 >100nd PA-KE >100 >100 >100 >100 nd PA-EKE 25 >100 100 >100 nd PA-KKEK 25100 25 100 nd Gentamycin 6.25 3.12 1.56 0.78 0.78 nd—not determined

As shown in Table 3, lipopeptides, which contain either short peptidesof all L-amino acid residues or short diastereomeric peptides, allhaving a positive charge that is greater than +1, exhibit antibacterialactivity.

TABLE 4 Minimal Inhibitory Concentration (μM) of the lipopeptides onbacteria growth. Conjugate S. aureus E. faecium Designation E. coli P.Aeruginosa A. Baumaannii E. cloacae E. faecalis S. aureus (MRSA) (VRE) 112.5 6.25 N.D. 3.125 3.125 6.25 6.25 N.D. 2 25 6.25 25 25 12.5 12.5 12.53.125 3 6.25 6.25 12.5 6.25 6.25 6.25 6.25 12.5 4 25 6.25 25 50 12.512.5 12.5 N.D. 5 6.25 6.25 6.25 25 12.5 12.5 12.5 N.D. 6 50 25 25 100 5050 N.D. N.D. 10 6.25 3.25 3.125 1.56 3.125 3.125 3.125 N.D. N.D. = Notdetermined

The results for lipopeptides 1-6, and 10, summarized in Table 4, revealthat the lipopeptides of the invention are potent against most bacteriaexamined. In addition, lipopeptides 2 and 10 are highly active againstVRE and MRSA, respectively, indicating that these bacteria are notresistant to the lipopeptides.

TABLE 5 Minimal Inhibitory Concentration (MIC) of the lipopeptides onbacteria growth. Minimal Inhibitory Concentration (μM) Bacteria Gram (+)Gram (−) E. aerogenes E. coli Conjugate S. aureus ATCC P. aeruginosaATCC Designation ATCC 6538P 35029 ATCC 27853 35218 [D]-L₆K₆ >50 >50 >5050 [D]-L₆K₆-DA 6.25 50 12.5 12.5 [D]-L₆K₆-DDA 12.5 50 25 25 [D]-L₆K₆-MA50 >50 50 50 [D]-L₆K₆-PA 50 >50 >50 50Results are the mean of 3 independent experiments, each performed induplicate.

As shown in Table 5, fatty acids such as DA or DDA improvedsignificantly the antibacterial activity of the diastereomeric peptide[D]-L₆K₆. Longer fatty acids such as MA or PA improved it slightly.

TABLE 6 Minimal inhibitory concentrations (μM) of the lipopeptides onbacteria S. aureus E. Coli P. Aeruginosa A. baumannii II Conjugate ATCCE. Coli ATCC ATCC ATCC Designation 25922 D21 27853 19606 6538PDDA-DL6R6-25 >100 50 50 >100 12.5 DDA-DL6H6-26 >100 >100 >100 100 100DDA-DL6R3H3- 100 100 100 100 100 27 DDA-DL6K6-22 75 25 25 25 3.125DDA-DL6K3H3- 100 50 50 50 18.75 28 Gentamycin 6.25 12.5 12.5 12.5 12.5

TABLE 7 Minimal inhibitory concentrations of the lipopeptides (μM) onbacteria growth at pH 5.5 A. baumannii S. aureus Conjugate E. Coli E.Coli P. Aeruginosa ATCC II Designation ATCC 25922 D21 ATCC 27853 19606ATCC 6538P DDA-DL6R6- 100 50 50 75 50 25 DDA-DL6H6- >100 100100 >100 >100 26 DDA- 100 100 100 >100 100 DL6R3H3-27 DDA-DL6K6- 50 2512.5 25 >100 22 DDA- >100 >100 >100 >100 >100 DL6K3H3-28 Gentamycin 12.512.5 12.5 12.5 25

As shown in Tables 6 and 7, the antibacterial activity exerted by thelipopeptides is dependent on the pH.

In order to evaluate the contribution of the fatty acids to theantibacterial activity of the lipopeptides of the invention, theantibacterial activity of the lipopeptides was compared to that of theparent peptides (without the conjugated fatty acid).

TABLE 8 Minimal Inhibitory Concentration (μM) of the lipopeptides onbacteria growth. Minimal Inhibitory Concentration (μM) Bacteria Gram (+)Gram (−) S. aureus E. coli Conjugate (ATCC B. subtilis P. aeruginosa A.baumannii (ATCC Designation 6538P) (ATCC 6051) (ATCC 27853) (ATCC 19606)25922) DK4G8 >100 >100 >100 >100 >100 PA-DK4G8-2 12.5 3.125 6.25 25 25DK4A8 >100 >100 >100 >100 >100 PA-DK4A8-5 12.5 3.125 6.25 6.25 6.25DK4V8 >100 60 >100 >100 >100 PA-DK4V8-7 >100 >100 >100 >100 >100

As shown in Table 8, conjugation of palmitic acid to peptides 2 and 5was associated with significantly enhanced antibacterial activity.

Example 4 Antifungal Activity of the Lipopeptides at pH 7.4 and pH 5.5

The antifungal activity of the lipopeptides was examined in sterile96-well plates (Nunc F96 microtiter plates) in a final volume of 200 μLas follows: 100 l of a suspension containing fungi at a concentration of1×10⁴ Colony-Forming Units (CFU)/ml in culture medium (RPMI 1640, 0.165M MOPS with L-glutamine, without NaHCO₃) at pH 7.4 or pH 5.5 (adjustmentof the pH was done by diluted solutions of HCl and NaOH) were added to100 μl of water containing the peptide in serial 2-fold dilutions inwater. The fungi were incubated in the presence of the lipopeptides for24-48 h at 35° C. in a Binder KB115 incubator under agitation. Growthinhibition was determined by measuring the absorbance at 620 nm with aMicroplate autoreader E1309 (Bio-Tek Instruments). Antifungal activityis expressed as the minimal inhibitory concentration (MIC), theconcentration at which 100% inhibition of fungi growth was observedafter the incubation time mentioned above. The fungi used were:Aspergillus niger ATCC 9642, Candida albicans ATCC 10231 andCryptococcus neoformans ATCC 66031. If not indicated otherwise, thefungi growth was detected at pH 7.4.

TABLE 9 Minimal Inhibitory Concentration (μM) of short lipopeptides onfungi growth. Minimal Inhibitory Concentration (μM) Yeast Fungi CandidaCyrptococcus Aspergillus Albicans neoformans Fomigatus Conjugate (ATCC10231) (ATCC MYA-422) (ATCC 26430) PA-KKKK 25 1.56 12.5 PA-KGGK 12.51.56 6.25 PA-KLLK 6.25 1.56 3.125 PA-KAAK 25 3.12 12.5 PA-K >1006.25 >100 PA-KK 12.5 2.5 6.25 PA-KKK 25 3.12 12.5 PA-KKK 25 3.12 12.5MYR-KKK >100 12.5 100 PA-EKKK 25 6.25 12.5 PA-RLLR 25 1.56 6.25PA-KE >100 >100 >100 PA-EKK 25 12.5 25 PA-KKEK 50 12.5 50 Amphotericin0.625 0.312 1.25Results are the mean of 3 independent experiments each performed induplicates, with standard deviation of 25%.

As shown in Table 9, lipopeptides, which contain all L-amino acid shortpeptides or short diastereomeric peptides having a positive charge thatis equal or greater than +1, exhibit antifungal activity.

TABLE 10 Minimal Inhibitory Concentration (μM) of lipopeptides on fungigrowth. Conjugate C. albicans C. neoformans A. fumigatus DesignationATCC 10231 ATCC 66031 ATCC 26430 1 3.125 6.25 6.25 2 3.125 6.25 12.5 33.125 3.125 6.25 4 3.125 1.56 6.25 5 12.5 1.56 100 6 3.125 1.56 12.5 103.125 1.56 6.25

The results for peptides 1-6 and 10 summarized in Table 10 show that allthe lipopeptides are highly active against all the fungi examined.

TABLE 11 Minimal Inhibitory Concentration (MIC) of the lipopeptides onfungi growth. Minimal Inhibitory Concentration (μM) Yeast Mould C.albicans C. neoformans A. fumigatus Peptide Designation ATCC 10231 ATCCMYA-422 ATCC 26430 [D]-L₆K₆ >50 >50 >50 [D]-L₆K₆-DA 25 1.56 12.5[D]-L₆K₆-DDA 12.5 0.78 6.25 [D]-L₆K₆-MA 1.56 0.78 1.56 [D]-L₆K₆-PA 1.560.78 1.56

Results are the mean of 3 independent experiments, each performed induplicates.

As shown in Table 11, conjugation of fatty acids having different lengthof aliphatic chain improved the antifungal activity of thediastereomeric peptide [D]-L₆K₆. The improvement of the antifungalactivity was more significant when the peptide was conjugated to fattyacid having longer aliphatic chain such as MA or PA.

TABLE 12 Minimal inhibitory concentrations of the lipopeptides (μM) onfungi growth. C. Neofor. A. Fumig. C. Albicans C. Neofor. A. Fumig. C.Albicans Conjugate Without Without Without With With With Designationserum serum serum serum serum serum DDA-DL6H6- 12.5 >100 >100 100100 >100 26 DDA- 3.125 25 12.5 25 100 >100 DL6R3H3-27 DDA- 1.56 25 12.56.25 100 >100 DL6K3H3-28 DDA-DL6R6- <0.78 3.125 3.125 <0.78 50 100 25DDA-DL6K6- <0.78 12.5 3.125 <0.78 50 100 22 Amphoterycin B <0.78 2.343.125 <0.78 <0.78 0.78

TABLE 13 Minimal inhibitory concentrations of the lipopeptides (μM) onfungi growth at pH 5.5. C. Neofor. A. Fumig. C. Albicans C. Neofor. A.Fumig. C. Albicans Conjugate Without Without Without With With WithDesignation serum serum serum serum serum serum DDA-DL6H6-26 3.125 6.259.375 50 100 >100 DDA-DL6R3H3- 1.56 6.25 25 25 100 >100 27 DDA-DL6K3H3-<0.78 12.5 12.5 12.5 100 >100 28 DDA-DL6R6-25 <0.78 6.25 12.5 2.34 50100 DDA-DL6K6-22 <0.78 75 100 1.56 100 >100 Amphoterycin B 3.125 3.1259.375 <0.78 <0.78 0.78

In order to evaluate the contribution of the fatty acid to theantifungal activity of the lipopeptides of the invention, the antifungalactivity of the lipopeptides was compared to that of the parentpeptides.

TABLE 14 Minimal Inhibitory Concentration (μM) of the lipopeptides onfungi growth. Minimal Inhibitory Concentration (μM) Yeast Fungi CandidaCryptococcus Aspergillus Aspergillus Aspergillus Conjugate albicansneoformans fumigatus flavus niger Designation (ATCC 10231) (ATCCMYA-422) (ATCC 26430) (ATCC 9643) (ATCC 9642)DK4G8 >100 >100 >100 >100 >100 PA-DK4G8-2 3.125 6.25 12.5 100 6.25DK4A8 >100 >100 >100 >100 >100 PA-DK4A8-5 12.5 1.56 100 80 10 DK4V8 >10050 >100 >100 >100 PA-DK4V8-7 3.125 3.125 >100 >100 >100

As shown in Table 14, the lipopeptides listed exert enhanced antifungalactivity compared to that of the parent peptides.

Thus, the group of 12-mer lipopeptides shown herein was significantlymore active against bacteria and fungi than their parent peptides, whichare not conjugated to fatty acids.

Example 5 Hemolytic Activity of the Lipopeptides

The effect of the lipopeptides of the invention on red blood cellhemolysis was next tested.

Fresh human red blood cells (hRBC) with EDTA were rinsed 3 times withPBS (35 mM phosphate buffer/0.15 M NaCl, pH 7.3) by centrifugation for10 min at 800 g and resuspended in PBS. Peptides dissolved in PBS werethen added to 50 μl of a solution of the stock hRBC in PBS to reach afinal volume of 100 μl (final erythrocyte concentration, 4% v/v). Theresulting suspension was incubated under agitation for 60 min at 3T. Thesamples were then centrifuged at 800×g for 10 min. Release of hemoglobinwas monitored by measuring the absorbance of the supernatant at 540 nm.Controls for zero hemolysis (blank) and 100% hemolysis consisted of hRBCsuspended in PBS and Triton 1%, respectively.

FIGS. 1-7 show the hemolytic effect of the lipopeptides of theinvention. Short lipopeptides containing 1 to 4 amino acid residues(FIGS. 1-4) and the 12-mer lipopeptides 2, 4, 5 and 6 (FIG. 5) do notexhibit significant hemolytic activity at concentrations lower than 12μM, at which concentrations that they exert specific antifungal andantibacterial activities. Similarly, lipopeptides 22, 26, and 28 do notexert hemolytic activity up to a concentration of 25 μM (FIGS. 6-7).

Example 6 Resistance of the Lipopeptides to Proteolytic Digestion

In order to reach their target, the lipopeptides have to withstandproteolytic digestion by proteases. Such degradation may occur from thetime the lipopeptides have been administered at one site till they reachtheir target site.

Equal amounts of the lipopeptides were dissolved in PBS (35 mM phosphatebuffer/0.15 M NaCl, pH 7.3) to yield a final concentration of 140 μM towhich 25 μM of either pepsin (from porcine stomach mucosa, Sigma),trypsin (from bovine pancreas, Sigma), or elastase (from humanleukocytes, Sigma) were added. The samples were incubated underagitation for 30 min at 37° C. After addition of the appropriateprotease inhibitor to stop the reaction, aliquots of the proteolyticdigest were injected to C₁₈ HPLC column and the amounts of the intactlipopeptide were evaluated using their absorbance at 215 nm.

TABLE 15 Proteolytic digestion (%) of the diastereomers. ConjugateDesignation Trypsin Pepsin Elastase 16 0 0 0 19 100 100 100

The results summarized in Table 15 show that lipopeptide 16 of theinvention is protected from proteolytic digestion by pepsin, trypsin,and elastase as assessed by reverse-phase HPLC. As a control,lipopeptide 19, which contains all L-amino acid residues with the samesequence as that of lipopeptide 16, was used. Thus, introducing D-aminoacids within the sequence of a particular lipopeptide significantlyprotects it from enzymatic degradation.

Example 7 The Anticancer Activity of the Lipopeptides

Prostate cancer cell lines (CL1 and 22RV1; ATCC, USA) were grown inRPMI-1640 supplemented with 10% FCS (Biological Industries, Beit Haemek,Israel). N1H-3T3 mouse fibroblast cell line (ATCC, USA) was grown inDMEM supplemented with 10% bovine serum. To test cytotoxicity againstthe cancer cells, aliquots of medium containing 1×10⁴ cells weredistributed into a 96-well plate (Falcon). After one day, the media werereplaced with 90 μl of fresh media and 10 μl of a solution containingdifferent concentrations of the peptides. The plate was then incubatedfor 24 h before adding to each well 50 μl of XTT reaction solution(Biological Industries, Beit Haemek, Israel); viability was determinedas previously described (Papo et al., (2003) J. Biol. Chem.278:21018-23). The LC50 (concentration at which 50% of the cells die)for each peptide was obtained from the dose-dependent cell viabilitycurves.

TABLE 16 LC50 of the lipopeptides (μM) against prostate cancer andnon-cancer cell lines. Conjugate Prostate cancer Prostate cancerDesignation 22RV1 CL1 3T3 DL6K6 >100 >100 >100 DDA-DL6K6- <3.125 <3.12550 22 MA-DL6K6-23 <3.125 <3.125 50 PA-DL6K6-24 <3.125 3.125 50 DL6R650 >100 >100 DDA-DL6R6 - 3.125 3.125 100 25

As shown in Table 16, coupling of a fatty acid to the diastereomericpeptides [D]L₆K₆ or [D]-L₆R₆ improved significantly the cytotoxic effectof the lipopeptides against cancer cells with almost no effect on normalcells.

In Vivo Studies with PC Xenografts

Subcutaneously (s.c.) implantation of human PC in mice is done asdescribed by Gavish Z, et al. (Prostate 2002; 51:73-83). Briefly, 0.1 mlAI CL1 and 22RV1 human PC cells (5×10⁶ cells) in Matrigel (BiologicalIndustries, Beit Haemek, Israel) is inoculated s.c. into the dorsal sideof five to six week-old nude male mice weighing 20-25 g (Harlen Co.,Israel). Two weeks after cell implantation, when the tumor diameterreaches 5 mm (this day is denoted day 1), the all L-amino acid peptideand its diastereomeric peptide (at 1 mg/kg, 0.1 mM), or vehicle (PBS,pH=7.4) are injected intratumorally (dosing volume of 2.5 ml/kg) threetimes a week for a total of 9 doses. Tumor size is measured by a caliperand recorded twice a week during a period of 28 days. Mice are weighedand tumor weight (mg) is estimated by using the formula oflength×width×depth×0.52 in mm³, assuming the specific gravity to be 1.At the end of the treatment, the mice are killed, and the tumors areremoved, photographed, and weighed. The animal experimentation arereviewed and approved by the Institutional Animal Care and UseCommittee.

Example 8 Antimicrobial Activity of Diastereomeric Magainin Conjugatedto a Fatty Acid

Previous studies indicated that coupling of a fatty acid to anantibacterial peptide, a magainin analog, resulted in endowing thepeptide with antifungal activity (18). It should be noted that thepeptide was devoid of antifungal activity before its coupling to thefatty acid.

In order to evaluate whether conjugation of a fatty acid to adiastereomeric magainin analog can endow the peptide with antifungalactivity, the magainin analog was synthesized as to contain four D-aminoacid residues. The sequence of the diastereomeric magainin analog is asfollows:

Gly-Ile-Gly-Lys-Phe-D-Leu-His-Ser-Ala-D-Lys-Lys-Trp-Gly-Lys-Ala-D-Phe-Val-Gly-D-Glu-Ile-Met-Asn- Ser-NH₂.

To the parent diastereomeric magainin analog, various fatty acids wereconjugated.

TABLE 17 Minimal Inhibitory Concentration (mM) of D-magainin analog onfungi and yeast growth. Minimal Inhibitory Concentration (μM) Yeast % ofCandida Cyrptococcus Fungi Hemolytic albicans neoformans Aspergillusactivity at Peptide (ATCC (ATCC fumigatus maximal Designation 10231)MYA-422) (ATCC 26430) MIC [D]4magainin >50 >50 >50 — [D]4magainin- >506.25 >50 2% UA [D]4magainin- >50 6.25 >50 8% PAResults are the mean of 3 independent experiments each performed induplicates, with standard deviation of 25%.

TABLE 18 Minimal Inhibitory Concentration (mM) of D-magainin analog onbacteria growth. Minimal Inhibitory Concentration (μM) Gram NegativeGram Positive E. coli B. subtilis Peptide ATCC P. aeruginosa ATCC S.aureus Designation 25922 ATCC 27853 6051 ATCC 6538P [D]4magainin >50 >5050 >50 [D]4magainin-UA >50 50 6.25 50 [D]4magainin-PA >50 >50 50 >50Results are the mean of 3 independent experiments each performed induplicates, with standard deviation of 25%.

The results in Tables 17 and 18 show that [D]-magainin is practicallynot active toward fungi and bacteria. Attachment of a fatty acid to theinactive [D]-magainin improved slightly its antibacterial activity i.e.,only against B. subtilis, and endowed it with antifungal activity onlyagainst C. neoformans (Tables 17 and 18). As the secondary structure of[D]-magainin was preserved compared to the secondary structure of theparent peptide, and as the conjugation of the fatty acids did not affect[D]-magainin structure, these results indicate that conjugation of afatty acid to a peptide having high hydrophobicity of its own cannotalways “rescue” an inactive peptide.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above. Rather the scope of the invention is defined bythe claims that follow.

1. A lipophilic conjugate comprising a peptide coupled to a fatty acid,the peptide, a cyclic analog or a salt thereof consisting of 2 to 14amino acid residues has a net positive charge that is equal or greaterthan +1 comprising at least two positively charged amino acid residues,said peptide prior to conjugation to the fatty acid being devoid of orhaving very weak antibacterial, antifungal and/or anticancer activity,wherein the peptide after conjugation to said fatty acid has at leastone activity selected from the group consisting of antibacterial,antifungal, and anticancer activity.
 2. The conjugate according to claim1, wherein the peptide is selected from all L-amino acid peptides, allD-amino acid peptides, and diastereomeric peptides.
 3. The conjugateaccording to claim 2, wherein the diastereomeric peptide comprises atleast one third amino acid residues in the D-configuration.
 4. Theconjugate according to claim 1, wherein the fatty acid is selected fromsaturated, unsaturated, monounsaturated and polyunsaturated fatty acids.5. The conjugate according to claim 1, wherein the fatty acid consistsof at least eight carbon atoms.
 6. The conjugate according to claim 5,wherein the fatty acid is selected from the group consisting of decanoicacid, undecanoic acid, dodecanoic acid, myristic acid, palmitic acid,stearic acid, arachidic acid, lignoceric acid, palmitoleic acid, oleicacid, linoleic acid, linolenic acid, arachidonic acid,trans-hexadecanoic acid, elaidic acid, lactobacillic acid,tuberculostearic acid, and cerebronic acid.
 7. The conjugate accordingto claim 1, wherein the peptide comprises a lysine di-peptide, lysinetri-peptide, or a lysine tetra-peptide.
 8. The conjugate according toclaim 1, wherein the peptide comprises at least two positively chargedamino acids selected from the group consisting of arginine, histidine,lysine, and combinations thereof and a hydrophobic amino acid selectedfrom the group consisting of leucine, valine, alanine, isoleucine,glycine, and a combination thereof.
 9. The conjugate according to claim8, wherein the peptide comprises at least two lysine residues and ahydrophobic amino acid selected from the group consisting of leucine,valine, alanine, isoleucine, and glycine.
 10. The conjugate according toclaim 8, wherein the peptide comprises leucine and at least twopositively charged amino acids selected from arginine or histidine. 11.The conjugate according to claim 8, wherein the peptide comprisesleucine and a combination of at least two positively charged amino acidsselected from the group consisting of lysine, arginine, and histidine.12. The conjugate according to claim 8, wherein the peptide comprises atleast two positively charged amino acids and a combination ofhydrophobic and non-hydrophobic amino acids.
 13. The conjugate accordingto claim 1, wherein the peptide comprises at least two positivelycharged amino acids and a negatively charged amino acid.
 14. Theconjugate according to claim 1, wherein the peptide is cyclic.
 15. Theconjugate according to claim 14 as set forth in SEQ ID NOS: 39 to 46.16. A pharmaceutical composition comprising as an active ingredient alipophilic conjugate according to claim
 1. 17. The pharmaceuticalcomposition according to claim 16, wherein the lipophilic conjugate asset forth in SEQ ID NO:1 to
 46. 18. The pharmaceutical compositionaccording to claim 16 formulated in a form of a solution, colloidaldispersion, cream, lotion, gel, foam, emulsion, spray, aerosol or anointment.
 19. A veterinary composition comprising a lipophilic conjugateaccording to claim 1 and formulated for veterinary use.
 20. A foodpreservative composition comprising a lipophilic conjugate according toclaim 1 for food preservation.
 21. A disinfecting composition comprisinga lipophilic conjugate according to claim 1 in an amount sufficient forsterilization.
 22. A method for treating a bacterial or fungal infectionin a subject comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising as an active ingredient a lipophilic conjugate according toclaim
 16. 23. The method according to claim 22, wherein the subject hasacne, poorly healing skin lesions, burn wounds or the infection isselected from fungal infections of the scalp, fungal infections relatedto traumatic wounds, bacterial infections related to traumatic wounds,and bacterial or fungal eye or ear infections.
 24. A method for treatingcancer in a subject comprising administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition according to claim 16.